Verotoxin (VT), also known as Shiga-like toxin (SLT), is known to cause bloody diarrhea and development of hemolytic uremic syndrome in Verotoxin Producing E. coli (VTEC) infection. One of the etiologic agents of VTEC infection is the virulent E. coli 0157. VT may also be produced by bacteria other than those that cause VTEC infection which may also result in a toxic syndrome in humans. In children or elderly adults with reduced immune responses, VT produced by bacteria growing in the intestines may get into the bloodstream by disrupting the intestinal epithelial cells. This may induce Hemolytic Uremic Syndrome (HUS) which is characterized by renal dysfunction and sometimes brain damage (see, e.g., Karmali, M. et al., The Lancet, 1, 619-620 (1983); Siegler, R., The Journal of Pediatrics, 125, 511-518 (1994)). To date, there exists no effective drug for these toxic syndromes. Antibiotics have not demonstrated efficacy to prevent the progression of the toxic syndromes (see, e.g., Carter, A. et al., The New England Journal of Medicine, 316, 1496-1500 (1987); Griffen, P. et al., Annals of Internal Medicine, 109, 705-712 (1988)). This might be because of the release of VT from bacteria killed by the antibiotics and the ineffectiveness of antibiotics against VT.
There are two types of Verotoxin, (or Shiga-like toxin) Verotoxin I (VT1 or SLT-1) and Verotoxin II (VT2 or SLT-2). (See, O'Brien et al., Curr. Top. Microbiol. Immunol., 180, 65-94. (1992)). VT2 producing E. coli have been isolated from patients suffering from VTEC infecion. (See, Russmann et al., J. Med. Microbiol., 40(5), 338-343 (1994)). Ostroff et al., J. Infect. Dis. 160, 994-998 (1989) and Kleanthous et al., Arch. Dis. child. 65, 722-727 (1990) report that E. coli 0157 strains that contained VT2 but not VT1 were more frequently associated with HUS. There are additional VT2 variants (VT2V) that have also been isolated clinically. (See, e.g., Microb. Pathog., 8, 47-60 (1990); FEBS Lett., 79, 27-30 (1991); Microb. Pathog., 5, 419-426 (1988); and J. Bacteriol., 170, 4223-4230 (1988)). Armstrong et al., J. Infect. Dis., 171(4), 1042-1045 (1995), have tested a VT absorbent in clinical trials, however, this drug works only in the intestine and is not available to absorb VT that has reached the bloodstream. A γ-globulin preparation showed very low neutralization activity for VT2 in comparison with that for VT1 (Ashkenazi, S. et al., The Journal of Pediatrics, 113, 1008-1014 (1988); Morooka, T. et al., Acta Paediatrica Japonica, 38, 294-295 (1996)). A mouse monoclonal antibody which neutralizes VT2 has been reported. However, this antibody was reported to show a relatively low binding affinity for VT2V (, Schmitt, C. et al., Infection and Immunity, 59, 1065-1073 (1991)).
Further, the use of murine monoclonal antibodies such as those described above have certain drawbacks in human treatment, particularly in repeated therapeutic regimens as explained below. Mouse monoclonal antibodies, for example, tend to have a short half-life in humans, and lack other important immunoglobulin functional characteristics when used in humans.
In addition, murine monoclonal antibodies contain substantial amino acid sequences that will be immunogenic when injected into a human patient. Numerous studies have shown that, after injection of a foreign antibody, the immune response elicited by a patient against the injected antibody can be quite strong, essentially eliminating the antibody's therapeutic utility after an initial treatment. Moreover, if mouse or other antigenic (to humans) monoclonal antibodies are used to treat various human diseases, subsequent treatments with unrelated mouse antibodies may be ineffective or even dangerous in themselves, because of cross-reactivity.
While the production of so-called “chimeric antibodies” (e.g., mouse variable regions joined to human constant regions) has proven somewhat successful, a significant immunogenicity problem remains. In general, the production of human immunoglobulins reactive with VT2 antigen with high affinity, as with many antigens, would be extremely difficult using typical human monoclonal antibody production techniques.
Thus, there is a need for improved forms of humanized immunoglobulins specific for VT2 antigen that are substantially non-immunogenic in humans, yet easily and economically produced in a manner suitable for therapeutic formulation and other uses. The present invention fulfills these and other needs.